Chlorophylls and bacteriochlorophylls, the ubiquitous pigments of photosynthesis, have been studied intensively in order to understand their photophysics and photochemistry (Scheer, 1991). Together with the more readily available but spectroscopically less informative porphyrins, they have also been used to gain a more general insight into energy and electron transfer, the mutual interactions of large aromatic molecules with central metals, and of the central metals with extra ligands.
Photosensitizers are of interest for utilization in photodynamic therapy (PDT) of tumors. This technique utilizes a combination of a non-toxic drug that absorbs light at a suitable wavelength with non-hazardous photosensitizing irradiation of the patient following administration of the drug.
Porphyrins have been shown to accumulate in tumor tissue and, upon irradiation of the tumor tissue, to absorb light in situ, providing a mean to detect tumors by location of the fluorescence. A crude derivative of hematoporphyrin, known as hematoporphyrin derivative or HPD, has been proposed both for detection and for photodynamic therapy of tumors. A form of HPD said to be more effective comprises a portion of HPD having an aggregate weight over 10 Kda and is the subject of U.S. Pat. No. 4,649,151. HPD or its active components have been described in U.S. Pat. No. 4,753,958 for topical treatment of skin diseases, and in Matthews et al., 1988, for sterilization of biological samples containing infectious organisms such as bacteria and virus. A mixture known as hematoporphyrin derivative (HPD) containing a high proportion of ether-linked hematoporphyrin (HP) oligomers is commercially available (Photofrin II, Quarda Logic Technologies Inc., Vancouver, BC, Canada).
In order to optimize the performance of the porphyrin drugs in therapeutics and diagnostics, several porphyrin derivatives have been proposed in which, for example, there is a central metal atom complexed to the four pyrrole rings, and/or the peripheral substituents of the pyrrole rings are modified and/or the macrocycle is dihydrogenated to Chl derivatives (chlorins) or tetrahydrogenated to BChl derivatives (bacteriochlorins).
Chlorophyll and bacteriochlorophyll derivatives have superior properties in comparison to porphyrins, but are less readily available and more difficult to handle. The potential of chlorophyll derivatives (Spikes and Bommer, 1991) and of bacteriochlorophyll derivatives (Beems et al., 1987; Dougherty, 1992; Fiedor et al., 1993; Kessel et al., 1993; Moser, 1998; Pandey et al., 1994; Tregub et al., 1993) for the diagnosis and therapy of cancer, has been studied. Due to their intense absorption in favorable spectral regions (650–850 nm) and their ready degradation after treatment, chlorophyll and bacteriochlorophyll derivatives have been identified as excellent sensitizers for PDT of tumors.
Complexes of cyclic tetrapyrroles with metals other than Mg were studied in the porphyrin and 17,18-dihydroporphyrin series to understand their spectrocospic and redox properties (Hynninen, 1991). Bacteriochlorophylls are of potential advantage compared to the chlorophylls because they show intense near-infrared bands, i.e. at considerably longer wavelengths than chlorophyll derivatives.
PCT International Application Publication No. WO 90/12573 to Dougherty describes derivatives of bacteriochlorophyll-a or -b or of the corresponding bacteriochlorins devoid of the central metal atom or in which the central metal atom may be a non-paramagnetic metal selected from Mg2+, Sn2+ and Zn2+, and the C-172-carboxyl group is esterified with a saturated or unsaturated hydrocarbyl residue of 8–25C, for the manufacture of a composition for use in a method to effect the destruction or impairment of undesired target biological substrates, which method comprises photosensitizing said substrate with an effective amount of said derivative, followed by irradiation of the target substrate with radiation in a wavelength band absorbed by said derivative for a time effective to impair or destroy the substrate. In addition, the compounds are said to be useful in photodynamic therapy and diagnostics. It is to be noted that although Sn2+ and Zn2+ complexes of bacteriochlorophyll-a or -b are claimed, these metal derivatives have not been exemplified nor was any method for their preparation described in the specification of said patent application WO 90/12573.
Under normal delivery conditions, i.e. in the presence of oxygen at room temperature and under normal light conditions, the BChl moieties are labile and have somewhat lower quantum yields for triplet state formation, when compared with, e.g., hematoporphyrin derivative (HPD). However, their possible initiation of biological redox reactions, favorable spectral characteristics and their ready degradation in vivo result in the potential superiority of bacteriochlorophylls over other compounds, e.g. porphyrins and chlorophylls, for PDT therapy and diagnostics and for killing of cells, viruses and bacteria in samples and in living tissue. Chemical modification of bacteriochlorophylls is expected to further improve their properties, but this has been very limited due to lack of suitable methods for the preparation of such modified bacteriochlorophylls (Hynninen, 1991).
European Patent Application published under No. 0584552 of the same applicant of the present application describes new conjugates of chlorophylls and bacteriochlorophylls at the C-173 position with amino acids, peptides and proteins for use in PDT therapy and diagnostics. The amino acid, peptide or protein residue is linked directly or via a spacer to the C-172-carboxyl group of the chlorophyll or bacteriochlorophyll molecule. These conjugates are prepared by methods which are mild enough to retain the acid-labile central Mg atom.
The C-132-carbomethoxy group of chlorophylls and bacteriochlorophylls is biosynthetically derived from the C-13 propionic acid side chain and part of the reactive β-ketoester system present in most chlorophylls at the isocyclic ring. However, unlike the C-17 propionic ester side chain, no methods have been available for either chemical or enzymatic transesterification at the C-133 position. The only reaction previously known for this group was its cleavage, leading to the 132-demethoxycarbonyl- or pyro-chlorophylls. German Patent Application No. DE 4121876 and PCT Publication No. WO 97/19081, both assigned to the present applicant, propose bacteriochlorophyll derivatives with modified ester residues at positions 133 and 173. However, these patent applications describe only bacteriochlorophyll derivatives with native methyl ester residues at position 133 and no methods for the preparation of other esters at position 133 were described therein.
It would be desirable to prepare new chlorophyll and bacteriochlorophyll derivatives for use in PDT, in order to maintain or even improve the favorable optical and physiological properties of Chls and BCbls while optimizing their photosensitizing potential as well as improving their chemical stability and optimizing their physiological lifetimes.